Trends in Molecular Medicine
ReviewMaking sense of nonsense GABAA receptor mutations associated with genetic epilepsies
Introduction
Genetic epilepsies refer to epilepsy syndromes previously classified as idiopathic generalized epilepsies (IGEs) (Box 1). IGEs affect approximately 1% of the population worldwide and are among the most common neurological disorders [1]. IGEs include several different epilepsy syndromes that vary in clinical severity from relatively benign febrile seizures (FS) and childhood absence epilepsy (CAE) to more severe juvenile myoclonic epilepsy (JME) and generalized epilepsy with febrile seizures plus (GEFS+). A subpopulation of GEFS+ patients have severe recurrent seizures with cognitive decline that has been referred to as severe myoclonic epilepsy in infancy (SMEI) or Dravet syndrome.
The majority of IGEs probably have a genetic component 2, 3 and it is becoming increasingly clear that mutations of transmembrane ion channels, including both voltage-gated and ligand-gated ion channels, are the underlying cause of many forms of human epilepsy, including those found in large pedigrees and in sporadic cases with de novo mutations (Box 1) 3, 4, 5. Mutation of ion channels that cause either a gain of function in excitatory neurotransmission or a loss of function in inhibitory neurotransmission could impair the balance between excitation and inhibition, leading to disinhibition and hyperexcitability in the brain. In this review we focus on the molecular pathophysiological basis for IGEs associated with inhibitory GABAA receptor channel subunit gene nonsense mutations that generate premature translation-termination codons (PTCs).
Section snippets
Genetic epilepsies and GABAA receptors
GABAA receptors are the primary mediators of fast inhibitory synaptic transmission in the central nervous system and have been repeatedly documented to play a critical role in animal models of seizures 6, 7, 8, 9, 10, 11, 12, 13. These inhibitory receptors are pentamers formed by assembly of multiple subunit subtypes (α1–α6, β1–β3, γ1–γ3, δ, ɛ, π, θ, and ρ1–ρ3) that form chloride ion channels and most commonly contain two α subunits, two β subunits and a γ or δ subunit. GABAA receptors mediate
Mutant mRNAs containing PTCs are subject to incomplete NMD
Recent studies have proposed a model of the pathogenesis of human genetic epilepsies associated with PTCs in GABAA receptor subunit genes 24, 25. The studies suggest that human epilepsy syndromes associated with PTCs are caused by a combination of degradation of unstable subunit mRNA and of unstable truncated subunit protein (Figure 2) 24, 25 and that this model of pathogenesis can be extended to several reported genetic epilepsies and other inherited disorders. In cells, transcription of
NMD efficiency is correlated with phenotype severity in other diseases
Studies of other diseases indicate that NMD efficiency is correlated with phenotype severity. Activation of NMD can rid cells of most mRNAs containing PTCs and reduce synthesis of truncated proteins that have potentially deleterious effects inside cells. This might reduce the manifestation of some genetic diseases if the wild-type allele is haplosufficient for physiological function or might only produce a mild phenotype compared to some C-terminal truncation mutations that have
Truncated mutant proteins are subject to ERAD, but at different rates
Because NMD is rarely complete, the remaining mutant transcripts should be translated and generate mutant protein. Similar to mRNA surveillance, at the protein level, trafficking deficient mutant subunits are subject to ER protein quality control leading to ER retention and/or ERAD after translation. Previous studies [25] have demonstrated that truncated mutant proteins translated from mutant mRNAs that escape NMD are often trafficking-deficient, misfolded and misrouted and consequently are
Subunit truncations produce loss-of-function alleles that are often trafficking-deficient
Studies 24, 25 on two different PTC-generating mutations in different GABAA receptor subunit genes demonstrated that both mutations caused a loss of subunit function. When expressed with partnering subunits in HEK 293T cells, mutant subunits had minimal expression on the cell surface compared with wild-type subunits. However, surface expression of the mutant subunits was not totally absent. Using whole-cell recording from wild-type and mutant α1β2γ2S receptors, it was demonstrated that very
Truncated GABAA receptor subunits are immature and subject to ERAD
Like all glycoproteins, GABAA receptor subunits are subject to ER quality control. Only correctly folded, assembled and mature subunits successfully oligomerize with other subunits and present on the surface and in synapses as pentamers (Figure 2). Previous work has demonstrated that cell surface GABAA receptor subunits are more mature and have a higher molecular mass compared with immature subunits inside the ER 24, 26. Studies have also demonstrated that both α1(975delC, S326fs328X) and
There are two categories of truncated GABAA receptor subunit proteins
The two truncated, mutant GABAA receptor α1(975delC, S326fs328X) [24] and γ2S(Q351X) [25] subunits had different effects on wild-type receptor channel functions. In cell systems in vitro, wild-type and mutant subunits were co-expressed to mimic the patient's autosomal dominant inheritance in which wild-type and mutant alleles coexist.
GABAA receptor profiles are altered when either γ2 or α1 subunits are reduced by PTCs
Studies of hemizygous control receptors and of the heterozygous γ2 subunit knockout mouse [47] both suggested that α1β2 receptors are formed and trafficked to the surface when there is reduced expression of γ2 subunits. This suggests that α1β2 receptors will be formed if γ2 subunits are reduced or unavailable, resulting in a small compensation for the loss of GABAAergic inhibition. In the presence of mutant γ2(Q351X) subunits, α1β2 receptors were formed at a reduced level compared to that
There is a destructive interaction between wild-type and mutant γ2(Q351X) subunits
The presence of mutant γ2(Q351X) subunits produced a dominant negative suppression of the biogenesis of wild-type γ2 subunits. It is likely that some other truncation mutations will have a similar dominant negative effect. Biochemical data indicated that the dominant negative effect of mutant subunits on wild-type subunits was probably due to oligomerization of mutant and wild-type subunits, resulting in ER retention and glycosylation arrest of both wild-type and mutant subunits. Retained
GABRA1+/– and GABRG2+/– mice do not adequately mirror loss-of-function epilepsy mutations
It is well established that impairment of GABAA receptor function leads to epilepsy and thus GABAA receptor subunit gene deletion mice have been created to study epilepsy. However, these heterozygous gene deletion knockout animals do not adequately mirror human loss-of-function epilepsy mutations. For example, homozygous GABRA1 gene deletion knockout mice only manifest tremor, whereas heterozygous mice are behaviorally normal [52]. By contrast, the human GABRA1 gene mutation 975delC, S326fs328X
Therapeutic implications
These recent studies of GABAA receptor epilepsy mutations provide molecular targets for potential new therapeutic strategies for the treatment of genetic epilepsies. Potential therapeutic approaches would include increasing wild-type and/or mutant GABAA receptor channel function or decreasing the disturbance of cellular signaling by the presence of the mutant GABAA receptor subunit protein. For GABAA receptor subunit mRNAs with PTCs that activate NMD and cause epilepsy due to loss of function
Concluding remarks
Recent research developments regarding GABAA receptor subunit nonsense mutations suggest potential novel therapeutic approaches for genetic epilepsies. Increasing the production of functional full-length protein and attenuating the production of nonfunctional truncated protein will probably be the most useful therapeutic approaches, in addition to using conventional antiepileptic drugs. As described above, recent studies suggest that ion channel PTC mutations can cause NMD and that neurons are
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